It is conventional practice in plant breeding to grow plants from seed of known parentage. Seed are planted in experimental plots, growth chambers, greenhouses, or other growing conditions and plants arising from the seed are either cross pollinated with other plants of known parentage or self-pollinated. The resulting seed are the offspring of the two parent plants or the self-pollinated plant, and are harvested, processed and planted to continue the plant breeding cycle. Specific laboratory or field-based tests may be performed on the plants, plant tissues, seed or seed tissues, in order to aid in the breeding process.
Generations of plants based on known crosses or self-pollinations are planted and then tested to see if these lines or varieties are moving towards characteristics that are desirable in the marketplace. Examples of desirable traits include, but are not limited to, increased yield, increased homozygosity, improved or newly conferred resistance and/or tolerance to specific herbicides and/or pests and pathogens, increased oil content, altered starch content, nutraceutical composition, drought tolerance, and specific morphological based trait enhancements.
As can be appreciated and as is well known in the art, these experiments can be massive in scale. They involve a huge labor force ranging from scientists to field staff to design, plant, maintain, and conduct the experiments, which can involve thousands or tens of thousands of individual plants. They also require substantial land resources. Plots or greenhouses can take up thousands of acres of land. Not only does this tie up large amounts of land for months while the plants germinate, grow, and produce seed, during which time they may be tested in the laboratory or field, but then the massive amounts of seed must be individually tagged, harvested and processed.
A further complication is that much of the experimentation goes for naught. It has been reported in the literature that some seed companies discard 80-90% of the plants early on in the experiment. Thus, much of the land, labor and material resources expended for growing, harvesting, and post-harvest processing ultimately are wasted for a large percentage of the seed.
Timing pressures are also a factor. Significant advances in plant breeding have put pressure on seed companies to quickly advance lines or varieties of plants that have more and better traits and characteristics. The plant breeders and associated workers are thus under increasing pressure to more efficiently and effectively process these generations and make significant selections early on in the breeding process.
Therefore, a movement towards earlier identification of traits of interest through laboratory based seed testing has emerged. Seed is non-destructively tested to derive genetic, biochemical or phenotypic information. If traits of interest are identified, the selected seed from specific plants are used either for further experiments and advancement, or to produce commercial quantities. Testing seed prevents the need to grow the seed into immature plants, which are then tested. This saves time, space, and effort. If effective, early identification of desirable traits in seed can lead to a great reduction in the amount of land needed for experimental testing, the amount of seed that must be tested, and the amount of time needed to derive the information necessary for making advancement decisions. For example, instead of thousands of acres of plantings and the subsequent handling and processing of all those plants, a fraction of acres and plants might be enough. However, because timing is still important, this is still a substantial task because even such a reduction involves processing, for example, thousands of seed per day.
A conventional method of attempting non-lethal seed testing is as follows: a single seed of interest is held with pliers above a sheet of paper laid out on a surface; a small drill bit is used to drill into a small location on the seed; debris is removed by the drill bit and collected on a sheet of paper; the paper is lifted; and the debris is transferred to a test tube or other container for subsequent laboratory analysis. This method is intended to be non-lethal to the seed. However, the process is slow, and its success and effectiveness depends heavily on the attention and accuracy of the worker. Each single seed must be manually picked up and held by the pliers. The drilling is also manual. Care must be taken with the drilling and the handling of the debris. Single containers, e.g. the individual test tubes, must then be handled and marked or otherwise tracked and identified. Additionally, the pliers and drill must be cleaned between the testing of each seed. There can be substantial risk of contamination by carry-over from seed to seed and the manual handling. Also, many times it is desirable to obtain seed material from a certain physiological tissue of the seed. For example, with corn seed, it may be desirable to genotype the endosperm. In such cases, it is not trivial, but rather is time-consuming and somewhat difficult, to manually grasp a small corn seed in such a way to allow the endosperm to be oriented to expose it for drilling. Testing other seed structures such as the seed germ is preferably avoided because removing material from such regions of the seed negatively impacts germination rates. Sometimes it is difficult to obtain a useful amount of material with this method.
Another example of non-lethally obtaining tissue from corn seed for laboratory analysis is disclosed at V. Sangtong, E. C. Mottel, M. J. Long, M. Lee, and M. P. Scott, Serial Extraction of Endosperm Drillings (SEED)—A Method for Detecting Transgenes and Proteins in Single Viable Maize Kernels, Plant Molecular Biology Reporter 19: 151-158, June 2001, which is incorporated by reference herein. It describes use of a hand-held rotary grinder to grind off particles, called “drillings,” from the kernel and collection of the particles to test for the presence of certain genes. However, this method also requires manual grasping and orientation of each individual seed relative to the grinder. It, too is time consuming and somewhat cumbersome. It also relies on the skill of the worker. This method raises issues of throughput, accuracy, whether a useful amount of material is obtained, and contamination. The grinder must be thoroughly cleaned between kernels in order to prevent contamination.
As evidenced by these examples, present conventional seed analysis methods used in genetic, biochemical, or phenotypic analysis, require at least a part of the seed to be removed and processed. In removing some seed tissue, various objectives may need to be met. These may include one or more of the following objectives:
(a) maintain seed viability after collection of seed tissue, if required.
(b) obtain at least a minimum required amount of tissue, without affecting viability.
(c) obtain tissue from a specific location on the seed, often requiring the ability to orient the seed in a specific position.
(d) maintain a particular throughput level for efficiency purposes.
(e) reduce or virtually eliminate contamination.
(f) allow for the tracking of separate tissues and their correlation to seeds from which the tissues were obtained.
(a) Viability
With regard to maintaining seed viability, it may be critical in some circumstances that the seed tissue removal method and apparatus not damage the seed in such a way that seed viability is reduced. It is often desirable that such analysis be non-lethal to the seed, or at least result in a substantial probability that the seed will germinate (e.g. no significant decrease in germination potential) so that it can be grown into a mature plant. For some analyses, seed viability does not need to be maintained, in which case larger quantities of tissue can often be taken. The need for seed viability will depend on the intended use of the seeds.
(b) Tissue Quantity
It is desirable to obtain a useful amount of tissue. To be useful, it must be above a certain minimum amount necessary in order to perform a given test and obtain a meaningful result. Different tests or assays require different quantities of tissue. It may be equally important to avoid taking too much tissue to avoid reducing germination potential of a seed, which may be undesirable. Therefore, it is desirable that the apparatus and methods for removing the seed tissue allow for variation in the amount of tissue taken from any given seed.
(c) Tissue Location
A useful amount of tissue also can involve tissue location accuracy. For example, in some applications the tissue must come only from a certain seed location or from specific tissue. Further, it is difficult to handle small particles like many seeds. It is also difficult to accurately position and orient seed. On a corn seed, for example, it may be important to test the endosperm tissue, and orient the corn seed for optimal removal of the endosperm tissue. Therefore, it is desirable that the apparatus and methods for removing the seed tissue are adapted to allow for location-specific removal, which may include specific seed orientation methods.
(d) Throughput
An apparatus and methodology for seed tissue removal must consider the throughput level that supports the required number of tissues to be taken in a time efficient manner. For example, some situations involve the potential need to test thousands, hundreds of thousands, or even millions of seed per year. Taking the hypothetical example of a million seed per year, and a 5-day work week, this would average nearly four thousand tests per day for each working day of a year. It is difficult to meet such demand with lower throughput methods. Accordingly, higher throughput, automatic or even semi-automatic methods for removal of seed tissue may be desirable.
(e) Avoiding Contamination
It is desirable that an apparatus and methodology for seed tissue removal not be prone to cross-contamination in order to maintain purity for subsequent analytical testing procedures. This can involve not only tissue location accuracy, such that tissue from a given location is not contaminated with tissue from a different location, but also methods involved in the removal and handling of the tissue to be tested, ensuring no contamination.
(f) Tracking Tissue to be Tested
Efficient processing of seeds and tissue removed from seeds presents a variety of challenges, especially when it is important to keep track of each seed, the tissue removed from such, and their correlation to each other, or to other tissues. Accordingly, it is desirable that apparatus and methods for tissue removal and testing allow for easy tracking of seed and tissue removed from such.
Conventional seed testing technologies do not address these requirements sufficiently, resulting in pressures on capital and labor resources, and thus illustrate the need for an improvement in the state of the art. The current methods are relatively low throughput, have substantial risk of cross-contamination, and tend to be inconsistent because of a reliance on significant manual handling, orienting, and removal of the tissue from the seed. This can affect the type of tissue taken from the seed and the likelihood that the seed will germinate. There is a need to eliminate the resources current methods require for cleaning between removal of individual portions of seed tissue. There is a need to reduce or minimize cross-contamination between unique tissue portions to be tested by carry-over or other reasons, or any contamination from any source of any other tissue. There is also a need for more reliability and accuracy. Accordingly, there is a need for methodologies and their corresponding apparatus which provide for seed tissue removal and testing that accomplishes one or more of the following objectives:
(a) maintains seed viability after seed tissue removal.
(b) obtains at least a minimum required amount of tissue, without affecting viability.
(c) obtains tissue from a specific location on the seed.
(d) maintains a particular throughput level for efficiency purposes.
(e) reduces or virtually eliminate contamination.
(f) allows for the tracking of separate tissues and their correlation to seeds from which the tissues were obtained.
Some of these objectives can be conflicting and even antagonistic. For example, obtaining a useful amount of tissue while maintaining seed viability requires taking some seed tissue, but not too much. Moreover, high-throughput methodologies involve rapid operations but may be accompanied by decreases in accuracy and increased risk of contamination, such that the methods must be done more slowly than is technically possible in order to overcome the limitations. These multiple objectives have therefore existed in the art and have not been satisfactorily addressed or balanced by the currently available methods and apparatuses. There is a need in the art to overcome the above-described types of problems such that the maximum number of objectives is realized in any given embodiment.